Chimeric adenoviral fiber protein and methods of using same

ABSTRACT

A recombinant adenovirus comprising a chimeric fiber protein and a therapeutic gene, a method of gene therapy involving the use of such an adenovirus, and an adenoviral transfer vector for the generation of such a recombinant adenovirus are provided.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a recombinant adenovirus comprising achimeric adenoviral fiber protein and the use of a recombinantadenovirus comprising a chimeric adenoviral fiber protein in genetherapy.

BACKGROUND OF THE INVENTION

Adenoviruses belong to the family Adenoviridae, which is divided intotwo genera, namely Mastadenovirus and Aviadenovirus. Adenoviruses arenonenveloped, regular icosahedrons 65-80 nm in diameter (Horne et al.,J. Mol. Biol., 1, 84-86 (1959)). The capsid is composed of 252capsomeres of which 240 are hexons and 12 are pentons (Ginsberg et al.,Virology, 28, 782-783 (1966)). The hexons and pentons are derived fromthree different viral polypeptides (Maizel et al., Virology, 36, 115-125(1968); Weber et al., Virology, 76, 709-724 (1977)). The hexon comprisesthree identical polypeptides of 967 amino acids each, namely polypeptideII (Roberts et al., Science, 232, 1148-1151 (1986)). The penton containsa penton base, which is bound to the capsid, and a fiber, which isnoncovalently bound to and projects from the penton base. The fiberprotein comprises three identical polypeptides of 582 amino acids each,namely polypeptide IV. The adenovirus serotype 2 (Ad2) penton baseprotein is an 8×9 nm ring-shaped complex composed of five identicalprotein subunits of 571 amino acids each, namely polypeptide III (Boudinet al., Virology, 92, 125-138 (1979)). Proteins IX, VI, and IIIa arealso present in the adenoviral coat and are thought to stabilize theviral capsid (Stewart et al., Cell, 67, 145-154 (1991); Stewart et al.,EMBO J., 12(7), 2589-2599 (1993)).

Once an adenovirus attaches to a cell, it undergoes receptor-mediatedinternalization into clathrin-coated endocytic vesicles of the cell(Svensson et al., J. Virol., 51, 687-694 (1984); Chardonnet et al.,Virology, 40, 462-477 (1970)). Virions entering the cell undergo astepwise disassembly in which many of the viral structural proteins areshed (Greber et al., Cell, 75, 477-486 (1993)). During the uncoatingprocess, the viral particles cause disruption of the cell endosome by apH-dependent mechanism (Fitzgerald et al., Cell, 32, 607-617 (1983)),which is still poorly understood. The viral particles are thentransported to the nuclear pore complex of the cell (Dales et al.,Virology, 56, 465-483 (1973)), where the viral genome enters thenucleus, thus initiating infection.

An adenovirus uses two separate cellular receptors, both of which mustbe present, to efficiently attach to and infect a cell (Wickham et al.,Cell, 73, 309-319 (1993)). First, the Ad2 fiber protein attaches thevirus to a cell by binding to an, as yet, unidentified receptor. Then,the penton base binds to α_(v) integrins, which are a family of aheterodimeric cell-surface receptors that mediate cellular adhesion tothe extracellular matrix molecules fibronectin, vitronectin, laminin,and collagen, as well as other molecules (Hynes, Cell, 69, 11-25(1992)), and play important roles in cell signaling processes, includingcalcium mobilization, protein phosphorylation, and cytoskeletalinteractions (Hynes, supra).

The fiber protein is a trimer (Devaux et al., J. Molec. Biol., 215,567-588 (1990)) consisting of a tail, a shaft, and a knob. The fibershaft region is composed of repeating 15 amino acid motifs, which arebelieved to form two alternating b-strands and b-bends (Green et al.,EMBO J., 2, 1357-1365 (1983)). The overall length of the fiber shaftregion and the number of 15 amino-acid repeats differ between adenoviralserotypes. For example, the Ad2 fiber shaft is 37 nm long and contains22 repeats, whereas the Ad3 fiber is 11 nm long and contains 6 repeats.The receptor binding domain of the fiber protein is localized in theknob region encoded by the last 200 amino acids of the protein (Henry etal., J. of Virology, 68(8), 5239-5246 (1994)). The regions necessary fortrimerization are also located in the knob region of the protein (Henryet al. (1994), supra). A deletion mutant lacking the last 40 amino acidsdoes not trimerize and also does not bind to penton base (Novelli et al.Virology, 185, 365-376 (1991)). Thus, trimerization of the fiber proteinis necessary for penton base binding. Nuclear localization signals thatdirect the protein to the nucleus to form viral particles following itssynthesis in the cytoplasm are located in the N-terminal region of theprotein (Novelli et al. (1991), supra). The fiber, together with thehexon, are the main antigenic determinants of the virus and alsodetermine the serotype specificity of the virus (Watson et al., J. Gen.Virol., 69, 525-535 (1988)). The fiber protein is glycosylated withsingle N-acetyl-glucosamine residues; however, the functionalsignificance of the glycosylation remains unclear (Caillet-Boudin etal., Eur. J. Biochem., 184, 205-211 (1989)).

Over ten fiber proteins from different adenoviral serotypes have beensequenced, only to reveal a larger sequence diversity than that observedamong other adenoviral proteins. For example, the knob regions of thefiber proteins from the closely related Ad2 and Ad5 serotypes are only63% similar at the amino acid level (Chroboczek et al., Virology, 186,280-285 (1992)), whereas their penton base sequences are 99% identical.Ad2 and Ad5 fiber proteins, however, both likely bind to the samecellular receptor, since they cross-block each other's binding. Incontrast, Ad2 and Ad3 fibers are only 20% identical (Signas et al., J.of Virology, 53, 672-678 (1985)) and presumably bind to differentreceptors, since each fails to cross-block the other's binding (Defer etal., J. of Virology, 64(8), 3661-3673 (1990)). Ad3 fiber utilizes sialicacid as its receptor, whereas Ad2 fiber does not. Pretreatment of cellswith neuraminidase or periodate abrogates Ad3, but not Ad2, binding.Also, soluble analogues of sialic acid block Ad3, but not Ad2, binding.However, sequence comparisons of the Ad2 and Ad3 fiber genes do showdistinct regions of conservation. Most of these regions are alsoconserved in the other human adenoviral fiber genes. Nonhuman adenoviralfiber genes show less homology to human serotypes but still trimerize.The receptors used by nonhuman serotypes are unknown.

Recombinant adenoviral vectors have been used for the cell-targetedtransfer of one or more recombinant genes to diseased cells or tissue inneed of treatment. Such vectors are characterized by the advantage ofnot requiring host cell proliferation for expression of adenoviralproteins (Horwitz et al., In Virology, Raven Press, New York, vol. 2,pp. 1679-1721 (1990); and Berkner, BioTechnigues, 6, 616 (1988)), and,if the targeted tissue for somatic gene therapy is the lung, thesevectors have the added advantage of being normally trophic for therespiratory epithelium (Straus, In Adenoviruses, Plenan Press, New York,pp. 451-496 (1984)).

Other advantages of adenoviruses as potential vectors for human genetherapy are as follows: (i) recombination is rare; (ii) there are noknown associations of human malignancies with adenoviral infectionsdespite common human infection with adenoviruses; (iii) the adenoviralgenome (which is a linear, double-stranded DNA) can be manipulated toaccommodate foreign genes that range in size; (iv) an adenoviral vectordoes not insert its DNA into the chromosome of a cell, so its effect isimpermanent and unlikely to interfere with the cell's normal function;(v) the adenovirus can infect non-dividing or terminally differentiatedcells, such as cells in the brain and lungs; and (vi) live adenovirus,having as an essential characteristic the ability to replicate, has beensafely used as a human vaccine (Horwitz et al. (1990), supra; Berkner etal. (1988), supra; Straus et al. (1984), supra; Chanock et al., JAMA,195, 151 (1966); Haj-Ahmad et al., J. Virol., 57, 267 (1986); and Ballayet al., EMBO, 4, 3861 (1985)).

A drawback to adenovirus-mediated gene therapy is that significantdecreases in gene expression are observed after two weeks followingadministration of the vector. In many therapeutic applications the lossof expression requires re-administration of the viral vector to overcomelosses in expression. However, following administration of the viralvector, neutralizing antibodies are raised against both the fiber andhexon proteins (Wohlfart, J. Virology, 62, 2321-2328 (1988); Wohlfart etal., J. Virology, 56, 896-903 (1985)). This antibody response againstthe virus then can prevent effective re-administration of the viralvector. Accordingly, recombinant adenoviral vectors capable of avoidingsuch neutralizing antibodies that would allow repeated doses ofadenoviral vectors to be administered in the context of gene therapywould represent a significant advance in current gene therapymethodology.

Another drawback of using recombinant adenovirus in gene therapy is thatall cells that express the aforementioned two receptors used byadenovirus to attach and infect a cell will internalize the gene(s)being administered--not just the cells in need of therapeutic treatment.Likewise, certain cells, such as lymphocytes, which lack the α_(v)integrin adenoviral receptors, are impaired in the uptake ofadenoviruses (Silver et al., Virology 165, 377-387 (1988); Horvath etal., J. of Virology, 62(1), 341-345 (1988)) and are not readily amenableto adenovirus-mediated gene delivery. Accordingly, limiting adenoviralentry to specific cells or tissues and/or expanding the repertoire ofcells amenable to adenovirus-mediated gene therapy would be asignificant improvement over the current technology. Targeted adenoviralgene delivery should expand the cells amenable to gene therapy, reducethe amount of adenoviral vector that is necessary to obtain geneexpression in the targeted cells, and reduce side effects andcomplications associated with increasing doses of adenovirus, such asinflammation and the transfection of normal, healthy cells.

Attempts have been made to target a virus to specific cells bysterically blocking adenoviral fiber protein with antibodies andchemically linking tissue-specific antibodies to the viral particle(Cotten et al., Proc. Natl. Acad. Sci. USA, 89, 6094-6098 (1992)).Although this approach has demonstrated the potential of targeted genedelivery, the complexity and reproducibility of this approach presentmajor hurdles blocking its application in clinical trials. Thedifficulties thus far encountered in targeting the virus by thesemethods involve the method of synthesis required, which is to make majoralterations in the viral particles following their purification. Thesealterations involve additional steps that covalently link largemolecules, such as polylysine, receptor ligands and antibodies, to thevirus (Cotten (1992), supra; Wagner et al., PNAS USA, 89, 6099-6103(1992)). The targeted particle complexes are not homogeneous instructure and their efficiency is sensitive to the relative ratios ofviral particles, linking molecules, and targeting molecules used.

The present invention seeks to overcome at least some of the aforesaidproblems of recombinant adenoviral gene therapy. It is an object of thepresent invention to provide recombinant adenoviral vectors capable ofavoiding neutralizing antibodies upon repeat administration, therebyenabling the maintenance of recombinant gene expression at atherapeutically effective level. It is another object of the presentinvention to provide a cell-specific/tissue-specific recombinantadenovirus so as to target gene therapy to selected cells/tissues,thereby reducing the amount of recombinant adenoviral vectoradministered and any side-effects/complications. A further object of thepresent invention is to provide means for modifying the viral particleat the level of gene expression, thus allowing viral particles to bepurified by conventional techniques. Another object of the presentinvention is to provide a method of gene therapy involving the use ofsuch a homogeneous adenovirus, without the need for additional chemicalmodifications of viral particles, such as psoralen inactivation, or theaddition of molecules to the virus which permit the covalent linkage ofadditional molecules to the virus. These and other objects andadvantages of the present invention, as well as additional inventivefeatures, will be apparent from the following detailed description.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a recombinant adenovirus comprising achimeric fiber protein, which differs from the native (wild-type) fiberprotein by the introduction of a nonnative amino acid sequence. Thenonnative amino acid sequence allows the adenovirus to be targetedtowards a protein, such as a receptor or a bi-or multi-specific protein,which is specific for binding to the nonnative amino acid sequence and atarget receptor, by facilitating direct binding between the nonnativeamino acid sequence and the protein, i.e., receptor or bi/multi-specificprotein. Alternatively, the nonnative amino acid sequence facilitatesproteolytic removal of the chimeric fiber protein to allow targeting ofthe adenovirus by means of another adenoviral coat protein, such as thepenton base. The present invention also provides an adenoviral transfervector, among others, comprising a recombinant fiber gene sequence forthe generation of a chimeric fiber protein, and a method of using aprotein-specific recombinant adenovirus, which is specific for a givenreceptor or bi-/multi-specific protein and which comprises a therapeuticgene, in gene therapy.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagram of the penton complex.

FIG. 2 is a partial restriction map of the vector pGBS.59-100.

FIG. 3 is a partial restriction map of the vector pl93 Ad5 Nde I/Sal I.

FIG. 4 is a partial restriction map of the vector pAcSG2.

FIG. 5 is a partial restriction map of the vector pl93 Ad5 FC (F-).

FIG. 6 is a partial restriction map of the vector pl93 FC (F2).

FIG. 7 is a partial restriction map of the vector pGBS.59-100 (F2).

FIG. 8 is a partial restriction map of the vector pAcSG2 (F2).

FIG. 9 is a partial restriction map of the vector pl93 FC (F3).

FIG. 10 is a partial restriction map of the vector pGBS.59-100 (F3).

FIG. 11 is a partial restriction map of the vector pAcSG2 (F3).

FIG. 12 is a partial restriction map of the vector pl93 FC (HSF:RGD).

FIG. 13 is a partial restriction map of the vector pGBS.59-100(HSF:RGD).

FIG. 14 is a partial restriction map of the vector pAcSG2 (HSF:RGD).

FIG. 15 is a diagram of the construction of the vectorpAd70-100dlE3.fiber7.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides, among other things, a recombinantadenovirus comprising a chimeric fiber protein. The chimeric fiberprotein comprises a nonnative amino acid sequence, in addition to or inplace of a native fiber amino acid sequence, which allows the adenovirusto bind to a protein, such as a receptor, which is other than a receptorbound by the native fiber, and which is referred to herein as a "targetreceptor," or a bi-/multi-specific protein, such as an antibody orfragment thereof, e.g., domain, with binding specificity for thenonnative amino acid sequence and for a target receptor. In the absenceof native fiber amino acid sequences that enable trimerization of thenative or chimeric fiber protein, the nonnative amino acid sequencecomprises one or more sequences that enable trimerization of thechimeric fiber protein, which preferably are not immediately adjacent tothe sequence that is specific for the aforesaid different protein, e.g.,target receptor or bi- or multi-specific protein. Alternatively, thechimeric fiber protein comprises a nonnative amino acid sequence, inaddition to or in place of a native fiber amino acid sequence, which isrecognized by a protease and is cleaved by the protease, effectivelyremoving the chimeric fiber protein and thereby allowing targeting ofthe adenovirus by means of another adenoviral coat protein, such as thepenton base.

By "nonnative amino acid sequence" is meant any amino acid sequence thatis not found in the native fiber of a given serotype of adenovirus andwhich is introduced into the fiber protein at the level of geneexpression. "Nonnative amino acid sequence" includes an amino acidsequence from an adenoviral serotype other than the serotype of theadenovirus with the chimeric fiber protein. (For example, an Ad3 fiberamino acid sequence or the entire Ad3 fiber expressed in an Ad5 chimericfiber protein or in place of an Ad5 fiber protein, respectively, is a"nonnative amino acid sequence.") It also includes a proteaserecognition sequence, i.e., a sequence that is recognized and cleaved bya protease.

By "protein-specific amino acid sequence" is meant any nonnative aminoacid sequence encoding a protein, protein domain or peptide, whetherspecifically bound by another protein or fragment thereof, and is meantto include an amino acid sequence that confers upon a chimeric fiber theability to directly bind to a target receptor or class of targetreceptors, preferably a cell-specific or tissue-specific receptor, andan amino acid sequence that confers upon a chimeric fiber the ability todirectly bind to a bi- or multi-specific protein, such as an antibody orfragment thereof, e.g., domain, which binds to a target receptor(s).

By "receptor" is meant a protein, including membrane-bound and solubleproteins, with high specific affinity for biologically activesubstances, such as hormones, antibodies, and enzymes.

By "chimeric fiber protein" is meant a fiber protein comprising anonnative amino acid sequence, which comprises either a protein bindingsequence or a protease recognition sequence, in addition to or in placeof a native fiber amino acid sequence, which comprises a protein bindingsequence. "Chimeric fiber protein" is intended to include a fiberprotein of a serotype which differs from that of the adenovirus on whichit is expressed, i.e., where the entire native fiber sequence isreplaced with an entire nonnative fiber sequence.

Incorporation of a protein-specific amino acid sequence into a chimericfiber molecule allows targeting through two or more separate proteinswhich are chemically or otherwise linked to make a bi- or multi-specificprotein. One component of the bi- or multi-specific protein binds to thefiber chimera. The second component or additional components of the bi-or multi-specific protein recognize(s) one or more additional targetreceptors. For example, a bi- or multi-specific protein can include abispecific multichain or single chain antibody (Cook et al., J. Immunol.Methods, 171, 227-237 (1994); Spooner et al., Human Pathol., 25, 606-614(1994)) in which one domain specifically binds an epitope on chimericfiber protein and the other domain specifically binds a target receptor.The bispecific antibodies bind to the chimeric fiber proteins in arecombinant adenovirus with the target receptor-specific domains of thebispecific antibodies available for binding to a target receptor.

Preferably, the entire native fiber protein or native receptor bindingsequence of the fiber protein has been replaced at the DNA level with anonnative protein-specific amino acid binding sequence. Alternatively,the native receptor binding sequence in the fiber gene has been renderedinactive at the DNA level by mutation of the sequence, such as byinsertional mutagenesis, for example, or rendered conformationallyinaccessible in the fiber protein, such as by insertion of a DNAsequence into or adjacent to the adenoviral fiber gene sequence, wherein"gene sequence" refers to the complete fiber gene sequence as well asany lesser gene sequence that is capable of being expressed as afunctional fiber protein. For insertional mutagenesis, the DNA sequenceis preferably inserted near the gene sequence encoding the nativereceptor binding sequence, so as to move the gene sequence encoding thenative receptor binding sequence within the fiber gene sequence suchthat, in the chimeric fiber protein, the native receptor bindingsequence is conformationally inaccessible for binding to a receptor. Inthe latter case, the inserted nonnative gene sequence that causes theconformational inaccessibility of the native receptor binding sequencein the fiber protein is preferably one that is specific for a targetreceptor or bi- or multi-specific protein. Such a recombinant adenoviruscan be used, for example, to study receptor binding, adenoviralattachment, and adenoviral infection in vitro or in vivo.

In a preferred embodiment of the present invention, the above-describedrecombinant adenovirus additionally comprises a gene or genes capable ofbeing expressed in a cell to which the virus has attached or by whichthe virus has been internalized and preferably is one having therapeuticutility, e.g., corrective DNA, i.e., DNA encoding a function that iseither absent or impaired, or a discrete killing agent, such as DNAencoding a cytotoxin that, for example, is active only intracellularly,or DNA encoding ribozymes or antisense molecules. Accordingly, the useof the term "therapeutic gene" is intended to encompass these and anyother embodiments of that which is more commonly referred to as genetherapy and is known to those of skill in the art. The recombinantadenovirus can be used for gene therapy or to study the effects ofexpression of the gene in a given cell or tissue in vitro or in vivo.

The recombinant adenovirus comprising a chimeric fiber protein and therecombinant adenovirus that additionally comprises a gene or genescapable of being expressed in a particular cell can be generated by useof a viral transfer vector, preferably an adenoviral transfer vector, inaccordance with the present invention. The viral transfer vector,preferably an adenoviral transfer vector, comprises a chimericadenoviral fiber gene sequence. The chimeric fiber gene sequencecomprises a nonnative gene sequence in place of a native fiber genesequence that encodes a receptor binding sequence, which has beendeleted, or in addition to a native receptor binding sequence, which hasbeen mutated or rendered conformationally expressed inaccessible in theexpressed chimeric fiber protein as described above. The nonnativesequence renders the adenovirus specific for binding to a protein, e.g.,a receptor or bi- or multi-specific protein, as described above and, inthe absence of native trimerization sequences, contains a sequence(s)which allows the chimeric protein to trimerize. Alternatively, thenonnative sequence comprises an entire fiber sequence from an adenovirusof a different serotype, which is then expressed in place of or inconjunction with native fiber on a given adenovirus. In other words,either all of the fibers on a given chimeric serotype or some of thefibers are of the native serotype, whereas others are of a nonnativeserotype. Another alternative is that the nonnative sequence comprisesone or more of a protease recognition sequence, which is cleaved by aprotease, thereby effecting removal of the chimeric fiber and targetingof the recombinant adenovirus by means of the penton base or other coatprotein (see FIG. 1 for diagram of penton complex). Based upon the highdegree of structural similarity between the fiber molecules of the morethan 41 human serotypes of adenovirus, it is expected that any one ofthe serotypes of human or nonhuman adenovirus may be used as the sourceof the fiber gene. It is preferred, however, that one of the serotypesfor which the fiber gene has been sequenced is used.

Restriction sites are introduced into the fiber gene sequence;preferably, such restriction sites are introduced into or flanking anative receptor binding sequence of the fiber gene sequence by asuitable method, such as PCR mutagenesis. Preferably, these restrictionsites are not already present in the fiber gene. Such sites facilitatethe removal or inactivation, such as by sequence alteration, of the DNAsequence encoding the native receptor binding sequence in a givenadenoviral genome, or the rendering of the native receptor bindingsequence conformationally inaccessible, thereby altering or eliminatingthe ability of the fiber to bind to a receptor normally bound by thefiber. A deleted native receptor binding sequence can be replaced with,or a mutated or conformationally inaccessible receptor binding sequencecan be accompanied by, a different DNA sequence, preferably a DNAsequence encoding specificity for binding to a protein, such as areceptor, preferably a cell-specific or tissue-specific receptor, orclass of receptors, or to a bi- or multi-specific protein withspecificity for a given receptor, for example. Unique restriction sitesin the fiber gene of one adenoviral serotype can be used to replaceregions of the native fiber gene with homologous regions of the fibergene from another serotype. Such restriction sites can even be used toreplace an entire native fiber sequence with a nonnative fiber sequence.

Preferably, the adenoviral vector is one into which any suitablenonnative amino acid sequence can be rapidly inserted. For example,unique Nde I and Bam HI restriction sites in p193 FC(F⁻) can be used toinsert receptor binding sequences from other fiber serotype genes.Alternatively, sequences also can be inserted into the fiber genesequence without the need for unique restriction sites through PCR.Because a recombinant adenovirus can be created via ligation ofrecombinant sequences with viral DNA or via homologous recombination,the adenoviral vector preferably has either (1) unique restriction sitesthat allow ligation of a vector fragment with the complementingfragments of the remaining viral genomes, as described in Example 1, or(2) adequate lengths of DNA on either side of the protein-specificsequence that allow efficient homologous recombination with viral DNA,as described in Example 1. A preferred adenoviral vector is shown inFIG. 10, which is a partial restriction map of such a vector. Theadenoviral vector of FIG. 10 was generated as described in Example 1.

DNA encoding short peptide sequences or protein domains capable ofbinding to a given protein, preferably a receptor or class of receptors,in particular cell- or tissue-specific receptor, is preferred forinsertion into the fiber gene sequence in which the native receptorbinding sequence has been deleted, mutated, or rendered conformationallyinaccessible. However, other DNA sequences, such as those that encodebi-/multi-specific protein recognition sequences, such asreceptor-specific antibody domains and sequences that encode antigenicepitopes recognized by specific antibodies, also may be used to replacethe native receptor binding sequence. The target receptor is optimallycell-specific or tissue-specific, and desirably is expressed only onthose cells or tissues to be treated.

A non-native, unique protease site also can be inserted into the fibergene sequence to target an adenovirus through the penton base or pentonbase chimeras. The protease site preferably does not affect fibertrimerization or receptor specificity of the fiber protein. The fiberchimera-containing particles are produced in standard cell lines, e.g.,those currently used for adenoviral vectors. Following production andpurification, the particles are rendered fiberless through digestion ofthe particles with a sequence-specific protease, which cleaves the fiberproteins and releases them from the viral particles to generatefiberless particles. For example, thrombin recognizes and cleaves at theamino acid sequence Val Pro Arg Gly Ser (TRINS) (SEQ ID NO: 8) (Stenfloet al., J. Biol. Chem., 257, 12280-12290 (1982)). Fiberless particleshave been shown to be stable and capable of binding and infecting cells(Falgout et al., J. of Virology, 62, 622-625 (1992)). These resultantparticles then can be targeted to specific tissues via the penton baseor other coat protein.

The size of the DNA used to replace the native receptor binding sequencemay be constrained, for example, by impeded folding of the fiber orimproper assembly of the penton base/fiber complex.

Alternatively, recombinant adenovirus comprising chimeric fiber proteinmay be produced by the removal of the native knob region, whichcomprises receptor-binding and trimerization domains, of the fiberprotein and its replacement with a nonnative trimerization domain and aprotein-specific binding domain (Peteranderl et al., Biochemistry, 31,12272-12276 (1992)). A recombinant adenovirus comprising a chimericfiber protein also may be produced by point mutation in the knob regionand the isolation of clones that are capable of trimerization butincapable of binding to the native receptor. In either case, and alsowith respect to the removal and replacement of the nativereceptor-specific binding sequence as described above, new proteinbinding domains may be added onto the C-terminus of the fiber protein orinto exposed loops of the fiber protein by inserting the nucleic acidsequence encoding the binding domain into the fiber gene sequence at theappropriate position.

Irrespective of which method is used to introduce a protein bindingsequence into the fiber protein, the fiber protein must be able totrimerize. If the fiber protein cannot trimerize, it will be unable tobind to penton base protein. Accordingly, the native receptor bindingsequence must be changed without affecting the ability of the moleculeto trimerize.

A recombinant chimeric fiber gene sequence can be moved from anadenoviral transfer vector into baculovirus or a suitable prokaryotic oreukaryotic expression vector for expression and evaluation of receptoror protein specificity and avidity, trimerization potential, penton basebinding, and other biochemical characteristics. Accordingly, the presentinvention also provides recombinant baculoviral and prokaryotic andeukaryotic expression vectors comprising a chimeric adenoviral fibergene sequence. The chimeric fiber gene sequence includes a nonnativesequence in addition to or in place of a native fiber amino acidsequence, which is specific for binding to a protein other than aprotein bound by the native fiber. The native fiber amino acid sequencemay be deleted, mutated, or rendered conformationally inaccessible asdescribed above with respect to the recombinant adenovirus comprising achimeric fiber protein. By moving the chimeric gene from an adenoviralvector to baculovirus or a prokaryotic or eukaryotic expression vector,high protein expression is achievable (approximately 5-50% of the totalprotein being the chimeric fiber). Accordingly, the present inventionalso provides a recombinant baculovirus comprising a chimeric fiber geneand a chimeric adenoviral fiber protein comprising a nonnative aminoacid sequence in addition to or in place of a native fiber amino acidsequence. The nonnative amino acid sequence is specific for binding to aprotein, such as a receptor or a bi-/multi-specific protein, or encodesa protease cleavage site as described above. For proteincharacterization studies, the recombinant chimeric fiber protein (rcFprotein, such as rcF5) can be purified using any suitable methods, suchas those described by Wickham et al. (1993), supra.

Various characteristic parameters of the fiber protein of interest canbe assessed. Specificity and affinity of the receptor or otherprotein/rcF interaction can be assessed by Scatchard analysis as shownpreviously by Wickham et al. (1993), supra, for wild-type penton baseprotein. Receptor specificity can be further assessed by usingantibodies and peptides specific for the targeted receptor to block rcF5binding to cells, using conventional methods. rcF binding to penton baseprotein can be assessed by its ability to precipitate radiolabeledpenton base protein when coupled to protein A-coated beads via anantibody to the fiber protein.

Viral attachment, entry and gene expression are evaluated initially byusing the adenoviral vector containing the insert of interest togenerate a recombinant virus expressing the chimeric fiber protein and amarker gene, such as β-galactosidase. β-galactosidase expression incells infected with adenovirus containing the β-galactosidase gene(Ad-LacZ) can be detected as early as two hours after adding Ad-Gluc tocells. This procedure provides a quick and efficient analysis of cellentry of the recombinant virus and gene expression, and is implementedreadily by an artisan of ordinary skill using conventional techniques.

A recombinant virus, which lacks a native receptor binding sequence inthe fiber, can be produced in human embryonic cell line 293 (HEK 293),which allows replication of Ad5LacZ virus in which the LacZ genereplaces the E1 region of the adenoviral genome. For producingrecombinant adenovirus containing chimeric fiber, the 293 cell line mustexpress the receptor to which the chimeric fiber protein is targeted. Inthe absence of constitutive receptor expression, the receptor gene canbe transfected into the 293 cell line to create a stably expressing cellline.

Recombinant adenoviruses of the present invention can be used to treatany one of a number of diseases by delivering to targeted cellscorrective DNA, i.e., DNA encoding a function that is either absent orimpaired, or a discrete killing agent, e.g., DNA encoding a cytotoxinthat, for example, is active only intracellularly. Diseases that arecandidates for such treatment include, for example, cancer, e.g.,melanoma, glioma or lung cancers; genetic disorders, e.g., cysticfibrosis, hemophilia or muscular dystrophy; pathogenic infections, e.g.,human immunodeficiency virus, tuberculosis or hepatitis; heart disease,e.g., preventing restenosis following angioplasty or promotingangiogenesis to reperfuse necrotic tissue; and autoimmune disorders,e.g., Crohn's disease, colitis or rheumatoid arthritis.

One skilled in the art will appreciate that suitable methods ofadministering a recombinant adenovirus of the present invention to ananimal for purposes of gene therapy (see, for example, Rosenfeld et al.,Science, 252, 431-434 (1991); Jaffe et al., Clin. Res., 39(2), 302A(1991); Rosenfeld et al., Clin. Res., 39(2), 311A (1991); Berkner,BioTechniques, 6, 616-629 (1988)), chemotherapy, and vaccination areavailable, and, although more than one route can be used to administersuch a recombinant adenovirus, a particular route can provide a moreimmediate and more effective reaction than another route.Pharmaceutically acceptable excipients are also well-known to those whoare skilled in the art, and are readily available. The choice ofexcipient will be determined in part by the particular method used toadminister the recombinant adenovirus. Accordingly, there is a widevariety of suitable formulations for use in the context of the presentinvention. The following methods and excipients are merely exemplary andare in no way limiting.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the compound dissolved indiluents, such as water, saline, or orange juice; (b) capsules, sachetsor tablets, each containing a predetermined amount of the activeingredient, as solids or granules; (c) suspensions in an appropriateliquid; and (d) suitable emulsions. Tablet forms can include one or moreof lactose, mannitol, corn starch, potato starch, microcrystallinecellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellosesodium, talc, magnesium stearate, stearic acid, and other excipients,colorants, diluents, buffering agents, moistening agents, preservatives,flavoring agents, and pharmacologically compatible excipients. Lozengeforms can comprise the active ingredient in a flavor, usually sucroseand acacia or tragacanth, as well as pastilles comprising the activeingredient in an inert base, such as gelatin and glycerin, or sucroseand acacia, emulsions, gels, and the like containing, in addition to theactive ingredient, such excipients as are known in the art.

The recombinant adenovirus of the present invention, alone or incombination with other suitable components, can be made into aerosolformulations to be administered via inhalation. These aerosolformulations can be placed into pressurized acceptable propellants, suchas dichlorodifluoromethane, propane, nitrogen, and the like. They mayalso be formulated as pharmaceuticals for non-pressured preparationssuch as in a nebulizer or an atomizer.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The formulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid excipient, for example, water, for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions can be prepared from sterile powders, granules, and tabletsof the kind previously described.

Additionally, the recombinant adenovirus of the present invention may bemade into suppositories by mixing with a variety of bases such asemulsifying bases or water-soluble bases.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams, or spray formulascontaining, in addition to the active ingredient, such carriers as areknown in the art to be appropriate.

The dose administered to an animal, particularly a human, in the contextof the present invention will vary with the gene of interest, thecomposition employed, the method of administration, and the particularsite and organism being treated. However, the dose should be sufficientto effect a therapeutic response.

In addition to the recombinant adenovirus of the present invention, therecombinant vectors, e.g., the adenoviral transfer vector, also haveutility in vitro. They can be used as a research tool in the study ofadenoviral attachment and infection of cells and in a method of assayingreceptor-ligand interaction. Similarly, the recombinant fiber proteincomprising a nonnative amino acid sequence in addition to or in place ofa native receptor binding sequence can be used in receptor-ligand assaysand as adhesion proteins in vitro or in vivo, for example.

The following examples further illustrate the present invention and, ofcourse, should not be construed as in any way limiting its scope.

EXAMPLE 1

This example describes how to change adenoviral antigenicity withoutchanging receptor specificity by creating a chimeric fiber protein inwhich the native Ad5 receptor binding domain is replaced with thenonnative Ad2 receptor binding domain.

The Ad2 fiber gene was amplified by PCR, wherein an Xho I site wasincorporated into the 5' end of the sense PCR primer of SEQ ID NO:1, andXma I and Bam HI sites were incorporated into the 5' end of theantisense primer of SEQ ID NO:2 to allow cloning into the Xho I/Xma Icloning sites in the vector pAcSG2 (FIG. 4) (Pharmingen, San Diego,Calif.) to create the vector pAcSG2 (F2) (FIG. 8). The pAcSG2 (F2) wasused to evaluate the fiber chimera at the protein level for receptor andpenton base binding activity.

The Nde I/Bam HI fragment of the fiber2 gene was removed from pAcSG2(F2) and cloned into the vector p193 FC (F-) (FIG. 5) to create p193 FC(F2) (FIG. 6). The vector pl93 FC (F-) was used as the base vector formaking all chimeric fiber adenoviruses. The p193 FC (F-) vector wascreated by cutting the p193 Ad5 (Nde I/Sal I) (FIG. 3) vector with Nde Iand Mun I to remove most of the Ad5 fiber gene, including its stop andpolyadenylation signals, and by replacing the Nde I/Mun I fragment witha synthetic oligonucleotide, which lacks the amino acid coding regionfor Ad5 fiber but retains the Ad5 fiber stop and polyadenylation signal.The synthetic oligonucleotide was prepared from two sense and antisensecomplementary oligonucleotides, SEQ ID NO:3 and SEQ ID NO:4,respectively, which recreate cut Nde I and Mun I sites when paired andcontain a Bam HI site just upstream of the stop codon to allowdirectional cloning into the Nde I/Bam HI sites. The Nde I/Sal Ifragment containing the chimeric Ad2/Ad5 fiber gene was then cloned intothe vector pGBS.59-100 (FIG. 2) to create the transfer vectorpGBS.59-100 (F2) (FIG. 7). The pGBS.59-100 (F2) transfer vector was thencut with Sal I, purified and transfected into an appropriate cell linewith a complementing 27,530 bp AdS DNA fragment (left arm, 0-27,530 bp)to create recombinant virus through homologous recombination. Anappropriate cell line is any cell line which expresses the receptor forthe chimeric fiber and which is capable of replicating the adenoviralvector. The complementing fragment of Ad5 DNA was prepared by cuttingthe Ad5 DNA with the restriction enzyme Srf I, which cuts the Ad5 genomeonce at position 27,530 in the wild-type Ad5 genome. The larger 27,530bp piece was then isolated from the smaller bp fragment using a CsClgradient, although an agarose gel or other appropriate separationtechnique could have been utilized.

Alternatively, viral DNA can be cut with a restriction enzyme, such asSpe I, which cuts at position 27,082 in the wild-type Ad5 genome. The27,082 bp Spe I fragment can be isolated from the smaller fragment asdescribed above and then ligated with the complementing Spe I/Sal Ifragment from the pGBS.59-100 (F2) vector and then transfected into theappropriate cell line. Recombinant virus then can be isolated by plaqueassay and verified as recombinant using PCR probes specific for thechimera and by restriction analysis.

EXAMPLE 2

This example describes how to change receptor specificity andantigenicity by creating a chimeric fiber protein in which the nativeAd5 receptor binding domain is replaced with the nonnative Ad3 receptorbinding domain. oligonucleotide primers were used to amplify a largefraction of the Ad3 fiber gene using PCR. The 5' sense primer of SEQ IDNO:5 contained an in-frame mutation that incorporated an Nde I site,whereas the antisense oligonucleotide of SEQ ID NO:6 incorporated a BamHI site to allow cloning of the amplified fragment into pAcSG2 (F2) inwhich the corresponding Nde I/Bam HI region of the Ad2 fiber gene wasremoved. The Nde I/Bam HI fragment of the gene for Ad3 fiber was thenremoved from the vector pAcSG2 (F3) (FIG. 11) and cloned into the vectorpl93 FC (F-) to create p193 FC (F3) (FIG. 9). The Nde I/Sal I fragmentcontaining the chimeric Ad3/Ad5 fiber gene was then cloned into thevector pGBS.59-100 to create the transfer vector pGBS.59-100 (F3) (FIG.10). The pGBS.59-100 (F3) transfer vector was then cut, purified, andtransfected into the appropriate cell line with Ad5 arms as inExample 1. Recombinant virus was then isolated and verified to berecombinant as in Example 1.

The receptor for Ad3 contains a sialic acid component, which is requiredfor binding of Ad3, while binding of Ad5 does not involve sialic acid.Since sialic acid is found on all higher eukaryotic cells, the Ad3/Ad5fiber chimera is capable of binding to all cells. Such a vector caninfect a broader range of cell types and exhibits different tissuespecificity than non-chimeric Ad5 vectors in vivo.

EXAMPLE 3

This example describes how receptor specificity can be changed andbinding domains can be incorporated at the C-terminus of mouseadenoviral fiber.

The fiber sequence from a nonhuman adenoviral serotype, mouse adenovirustype 1, for example, is amplified using PCR. Nhe I and Bam HI sitesincorporated into the sense and antisense PCR primers, respectively,allow subsequent cloning of the PCR product. The Nhe I site correspondsto a naturally occurring site in Ad5 fiber that occurs after thesequence encoding penton base recognition domains. The antisense primer,in addition to the required Bam HI site, contains a sequence encoding anα_(v) β₃ -specific RGD peptide following an amino acid spacer of 5-30amino acids (such as poly Ala Ser! or poly Gly!). A unique restrictionsite is incorporated into the sequence following the spacer sequence andthen again before the stop codon. The site allows the incorporation ofreceptor-specific sequences other than the α_(v) β₃ -specific RGDpeptide. The resultant PCR product is then cloned into pAcSG2 (F5) toreplace the corresponding Ad5 fiber sequence and create pAcSG2(MouseRGD). The Nde I/Bam HI fragment containing the chimeric fiber geneis cloned into pl93 FC (F-) to create p193 FC (MouseRGD). The Nde I/SalI fragment from pl93 FC (MouseRGD) is cloned into pGBS.59-100 to createthe transfer vector pGBS.59-100 (MouseRGD). The transfer vector is thenprepared and transfected along with complementing Ad5 DNA into cellsexpressing the α_(v) β₃ receptor as described in Example 1. Recombinantvirus containing the chimeric fiber gene is analyzed as in Example 1.Using the unique restriction site incorporated into the vector, otherreceptor binding domains, such as the P-selectin binding domain or asingle chain receptor-specific antibody, can be directly cloned into thevector. However, the cell line used for transfection must express thetargeted receptor in order for the recombinant virus to attach andinfect cells. Incorporation of receptor or antibody binding domains intofiber molecules that do not recognize human receptors allow for thetargeting of a vector using such a fiber without retaining residualamino acid sequences that recognize human receptors and preventefficient targeting.

EXAMPLE 4

This example describes how to change receptor specificity by mutating anative fiber receptor-binding domain and incorporating a nonnativebinding domain at the C-terminus or within an exposed loop of a mutantAd5.

A mutated fiber gene, one which generates fiber that can trimerize butcannot bind to a native fiber receptor, is amplified by PCR usingprimers that incorporate proper restriction sites for cloning. Theantisense primer, in addition to the required Bam HI site, contains asequence encoding an α_(v) β₃ -specific RGD peptide following an aminoacid spacer of 5-30 amino acids, such as poly (Ala Ser) or poly Gly. Aunique restriction site is incorporated into the sequence following thespacer sequence and then before the stop codon. The site allows theincorporation of receptor-specific sequences other than the α_(v) β₃-specific RGD peptide. The amplified chimeric gene is cloned into thepl93 FC (F-) plasmid to obtain p193 FC (F5*:β3). The Nde I/Sal Ifragment containing the chimeric fiber gene is then cloned into thepGBS.59-100 vector to obtain pGBS.59-100 (F5*:β3). The transfer vectoris prepared and transfected with complementing Ad5 DNA as described inExample 1. Recombinant virus containing the chimeric fiber gene isanalyzed as in Example 1. Other receptor-specific or antibody-specificbinding domains can be cloned into the vector to create fiber chimeraswith such sequences at the C-terminus of the protein or within exposedloops of the fiber molecule for targeting to other receptors orantibodies, respectively, as described in Example 3.

EXAMPLE 5

This example describes replacement of a knob with a trimerization domainand the incorporation of a binding domain at the C-terminus of the knobprotein.

The adenovirus type 2 fiber gene was amplified using PCR from Ad2 viralDNA and cloned into the baculovirus transfer vector, pBlueBac2(Invitrogen, La Jolla, Calif.), to generate the vector pBB2F. The uniquerestriction sites Pst I and Bam HI encompass the region of the fiber2gene encoding the knob region of the protein. These sites were used toremove the Pst I to Bam HI portion of the fiber gene and to replace itwith DNA encoding the trimerization domain from the heat shock factor(HSF) protein of K. lactis fused via a glycine spacer to an RGD peptidespecific for the integrin avb3. The DNA encoding the HSF domain and RGDpeptide was obtained through PCR from a plasmid containing the sequencefor the entire K. lactis HSF protein. The DNA sequence encoding the RGDpeptide was incorporated into the antisense DNA primer of SEQ ID NO:7used in the PCR of the HSF trimerization domain to create the DNAsequence encoding the HSF:RGD fusion protein. The sense primer containeda Pst I site native to the Ad2 fiber gene. The PCR product was thendigested with Pst I and Bam HI and cloned into the pAcSG2 (F2) vector toobtain the plasmid pAcSG2:HSF:RGD (FIG. 14). Unique Spe I and Sca Isites were incorporated into the chimeric HSF:RGD gene so that differentreceptor-specific or antibody-specific sequences could be rapidlyinserted into the gene in place of the RGD-coding sequence at the end ofthe sequence encoding the glycine spacer arm. The pAcSG2:HSF:RGD plasmid(FIG. 14) was used to make recombinant baculovirus which expresses thefusion protein at high levels. The fusion protein expressed was thecorrect size and formed a trimer. The Nde I/Bam HI fragment of thechimeric gene was then removed from the vector pAcSG2 (HSF:RGD) andcloned into the vector p193 FC (F-) to create p193 FC (HSF:RGD) (FIG.12). The Nde I/Sal I fragment containing the chimeric fiber gene wascloned into the vector pGBS.59-100 (FIG. 2) to create the transfervector pGBS.59-100 (HSF:RGD) (FIG. 13). The pGBS.59-100 (HSF:RGD)transfer vector was then cut, purified and transfected into theappropriate cell line with Ad5 arms as in Example 1. Recombinant viruswas then isolated and verified to be recombinant as in Example 1.

EXAMPLE 6

This example describes how to replace a knob with a trimerization domainand how to incorporate a binding domain containing a protease cleavagesite at the C-terminus of the knob.

A chimeric fiber can be targeted to a new receptor by incorporating anepitope into the chimera which is recognized by a bi-specific antibody.An additional RGD domain is incorporated at the C-terminus of theprotein and separated from the antibody epitope by a unique proteaserecognition site. The chimeric virus is capable of growing in tissueculture cells that express the receptor for the RGD sequence. Finalpreparations of virus are then exposed to the protease to remove the RGDsequence, leaving the epitope. The viral particles are then exposed to abi-specific antibody in which one half of the molecule recognizes theepitope on the chimeric fiber and the other half recognizes any desiredreceptor, e.g., cell- or tissue-specific, receptor. The RGD sequence isabsent and the virus binds to and enters only those cells recognized bythe bi-specific antibody.

EXAMPLE 7

This example describes how to change the adenoviral antigenicity andreceptor specificity of an Ad5 virus by replacing native Ad5 fiber withnonnative Ad7 fiber and demonstrates the ability of such recombinantvirus to infect cells in vitro and in vivo.

The Ad5 virus-Ad7 fiber construct was generated as shown in FIG. 15. Anapproximately 2.7 kb (Ad5 28689-31317 bp) fragment in pAd70-100 wasreplaced with a Pac I linker (pAd70-100dlE3.Pac). A Bam HI linker wasinserted at a unique Mun I site as indicated in FIG. 13 to producepAd70-100dlE3.Pac.Bam. A PCR-amplified Pac I-Bam HI fragment ofapproximately 1.1 kb containing the Ad7 fiber gene was inserted intopAd70-100dlE3.Pac.Bam to produce pAd70-100dlE3.fiber7.

In order to assess the ability of Ad5 virus with Ad7 fiber to infectcells in vitro and in vivo, reporter gene assays were performed. Areplication-defective recombinant adenoviral reporter vector designatedAdCMV-CATNeo was used in the reporter gene assay. The reporter vectorconsists of the adenoviral origin of replication and viral packagingsequences, a combination of strong eukaryotic promoter (cytomegalovirusor CMV-1) and splicing elements, the bacterial chloramphenicol acetyltransferase (CAT) gene sequence, the mouse β^(maj) -globin poly(A) site,the neomycin gene sequence (Neo), and sufficient adenoviral DNA to allowfor overlap recombination.

The reporter vector was used to generate AdCMV-CATNeo, AdCMV-CATNeo-dlE3(AdCMV-CATNeo+pAd70-100dlE3) and AdCMV-CATNeo-dlE3-Fiber7(AdCMV-CATNeo+pAd70-100dlE3.Fiber7) viruses. Each virus was grown inlarge scale, i.e., a 1 1 suspension of human embryonic kidney 293 cells,to yield virus at a concentration of 10¹² particles/ml. A549 cells wereinfected with an estimated 100, 300 or 1,000 particles/cell of one ofthe three viruses. After 48 hr, the cells were harvested and lysateswere prepared as described in Kass-Eisler et al., PNAS USA, 90,11498-11502 (December 1993). Using 50 μl of each lysate, CAT assays wereperformed and acetylated chloramphenicol products were separated by thinlayer chromatography using chloroform:methanol (95:5). The results ofthe assays indicated that each virus was able to infect cells andexpress gene products at appropriate levels. Accordingly, the virus inwhich the native fiber was replaced with a nonnative fiber could infectcells and express genes like the parental virus.

Following this study, adult Sprague Dawley rats were infected with 10⁸viral particles by direct cardiac injection as described in Kass-Eisleret al., supra. Five days later, the rats were sacrificed, cardiaclysates were prepared, and CAT assays were performed. The amount of theCAT gene product produced was compared between the dlE3 and dlE3-Fiber 7viruses. Results indicated that both viruses were able to infect cellsin vivo. The replacement of the wild-type Ad5 fiber gene with that ofAd7 did not impair the ability of the virus to infect cells.Accordingly, the virus in which the native fiber was replaced with anonnative fiber could also infect cells and express genes like theparental virus in vivo. These results support the utility of adenoviruswith chimeric fiber in the context of gene therapy.

All publications cited herein are hereby incorporated by reference tothe same extent as if each publication were individually andspecifically indicated to be incorporated by reference and were setforth in its entirety herein.

While this invention has been described with emphasis upon preferredembodiments, it will be obvious to those of ordinary skill in the artthat the preferred embodiments may be varied. It is intended that theinvention may be practiced otherwise than as specifically describedherein. Accordingly, this invention includes all modificationsencompassed within the spirit and scope of the appended claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 18                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA (synthetic)                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GCCGCTCGAGTTGCAGATGAAACGCGCCAGA31                                             (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA (synthetic)                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       AGGGCCCGGGAGGATCCTTATTCTTGGGCAATGTA35                                         (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 55 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA (synthetic)                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       TATGGAGGATCCAATAAAGAATCGTTTGTGTTATGTTTCAACGTGTTTATTTTTC55                     (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 57 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA (synthetic)                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       AATTGAAAAATAAACACGTTGAAACATAACACAAACGATTCTTTATTGGATCCTCCA57                   (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA (synthetic)                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       AACCCGGTGTACCCATATGATGAAAGCAGCTC32                                            (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA (synthetic)                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       AATGGATCCTCAGTCATCTTCTCTAATATAGGAAA35                                         (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 79 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: DNA (synthetic)                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       ATGGATCCAGTACTTTAATTGCGAATGTCTCCGCGTCCAAAACTAGTTCCACCTCCACCT60                CCGAGTTCATGGATCAAAT79                                                         (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       ValProArgGlySer                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 54 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..54                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       ATGAAGCGCGCAAGACCGTCTGAAGATACCTTCAACCCCGTGTATCCA48                            MetLysArgAlaArgProSerGluAspThrPheAsnProValTyrPro                              151015                                                                        TATGAC54                                                                      TyrAsp                                                                        (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      MetLysArgAlaArgProSerGluAspThrPheAsnProValTyrPro                              151015                                                                        TyrAsp                                                                        (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 55 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..9                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      GCCCAAGAATAAAGAATCGTTTGTGTTATGTTTCAACGTGTTTATTTTTCAATTG55                     AlaGlnGlu                                                                     (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 3 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      AlaGlnGlu                                                                     1                                                                             (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 62 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      CATATGGAGGATCCAATAAAGAATCGTTTGTGTTATGTTTCAACGTGTTTATTTTTCAAT60                TG62                                                                          (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 86 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..45                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      GGAGGTGGAGGTGGAACTAGTTTTGGACGCGGAGACATTCGCAAT45                               GlyGlyGlyGlyGlyThrSerPheGlyArgGlyAspIleArgAsn                                 151015                                                                        TAAAGTACTGGATTCATGACTCTAGACTTAATTAAGGATCC86                                   (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      GlyGlyGlyGlyGlyThrSerPheGlyArgGlyAspIleArgAsn                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 98 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..51                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      GAACTCGGAGGTGGAGGTGGAACTAGTTTTGGACGCGGAGACATTCGC48                            GluLeuGlyGlyGlyGlyGlyThrSerPheGlyArgGlyAspIleArg                              151015                                                                        AATTAAAGTACTGGATTCATGACTCTAGACTTAATTAAGGATCCAATAAA98                          Asn                                                                           (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      GluLeuGlyGlyGlyGlyGlyThrSerPheGlyArgGlyAspIleArg                              151015                                                                        Asn                                                                           (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..45                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      GGAGGTGGAGGTGGAACTAGTTTTGGACGCGGAGACATTCGCAAT45                               GlyGlyGlyGlyGlyThrSerPheGlyArgGlyAspIleArgAsn                                 151015                                                                        __________________________________________________________________________

What is claimed is:
 1. A chimeric adenovirus fiber protein whichcomprises a nonnative amino acid sequence in addition to a native fiberamino acid sequence, wherein said nonnative amino acid sequence is aprotease recognition sequence or a protein binding sequence selectedfrom the group consisting of a protein binding sequence from a serotypeof adenovirus that differs from the serotype of the native fiber aminoacid sequence, a bispecific protein binding sequence, and amultispecific protein binding sequence.
 2. The chimeric adenovirus fiberprotein of claim 1, wherein said native fiber amino acid sequence is aprotein binding sequence that has been moved within the fiber protein.3. The chimeric adenovirus fiber protein of claim 1, wherein saidnonnative amino acid sequence is located internally in the chimericadenovirus fiber protein.
 4. A recombinant adenovirus comprising thechimeric adenovirus fiber protein of claim
 3. 5. An isolated andpurified nucleic acid that encodes the recombinant adenovirus of claim4.
 6. A method of genetically modifying a cell in vitro which comprisesintroducing into said cell the recombinant adenovirus of claim
 4. 7. Anisolated and purified nucleic acid that encodes the chimeric adenovirusfiber protein of claim
 3. 8. A viral transfer vector comprising thenucleic acid of claim
 7. 9. A vector comprising the nucleic acid ofclaim 7, wherein said vector is selected from the group consisting of aprokaryotic expression vector, a eukaryotic expression vector, and arecombinant baculovirus.
 10. The chimeric adenovirus fiber protein ofclaim 1, wherein said nonnative amino acid sequence is in an exposedloop of the chimeric adenovirus fiber protein.
 11. A recombinantadenovirus comprising the chimeric adenovirus fiber protein of claim 1.12. An isolated and purified nucleic acid that encodes the recombinantadenovirus of claim
 11. 13. A method of genetically modifying a cell invitro which comprises introducing into said cell the recombinantadenovirus of claim
 11. 14. An isolated and purified nucleic acid thatencodes the chimeric adenovirus fiber protein of claim
 1. 15. A viraltransfer vector comprising the nucleic acid of claim
 14. 16. A vectorcomprising the nucleic acid of claim 14, wherein said vector is selectedfrom the group consisting of a prokaryotic expression vector, aeukaryotic expression vector, and a recombinant baculovirus.
 17. Achimeric adenovirus fiber protein which comprises a nonnative amino acidsequence in place of a native fiber amino acid sequence, wherein saidnonnative amino acid sequence is a protease recognition sequence or aprotein binding sequence selected from the group consisting of a proteinbinding sequence from a serotype of adenovirus that differs from theserotype of the native fiber amino acid sequence, a bispecific proteinbinding sequence, and a multispecific protein binding sequence.
 18. Thechimeric adenovirus fiber protein of claim 17, wherein said native fiberamino acid sequence is a protein binding sequence.
 19. The chimericadenovirus fiber protein of claim 17, wherein said nonnative amino acidsequence is located internally in the chimeric adenovirus fiber protein.20. A recombinant adenovirus comprising the chimeric adenovirus fiberprotein of claim
 19. 21. An isolated and purified nucleic acid thatencodes the recombinant adenovirus of claim
 20. 22. A method ofgenetically modifying a cell in vitro which comprises introducing intosaid cell the recombinant adenovirus of claim
 20. 23. An isolated andpurified nucleic acid that encodes the chimeric adenovirus fiber proteinof claim
 19. 24. A viral transfer vector comprising the nucleic acid ofclaim
 23. 25. A vector comprising the nucleic acid of claim 23, whereinsaid vector is selected from the group consisting of a prokaryoticexpression vector, a eukaryotic expression vector, and a recombinantbaculovirus.
 26. The chimeric adenovirus fiber protein of claim 17,wherein said nonnative amino acid sequence is in an exposed loop of thechimeric adenovirus fiber protein.
 27. A recombinant adenoviruscomprising the chimeric adenovirus fiber protein of claim
 17. 28. Anisolated and purified nucleic acid that encodes the recombinantadenovirus of claim
 27. 29. A method of genetically modifying a cell invitro which comprises introducing into said cell the recombinantadenovirus of claim
 27. 30. A recombinant adenovirus comprising thechimeric adenovirus fiber protein of claim 17, wherein said native fibersequence is replaced in its entirety with said nonnative fiber sequence.31. An isolated and purified nucleic acid that encodes the recombinantadenovirus of claim
 30. 32. A method of genetically modifying a cell invitro which comprises introducing into said cell the recombinantadenovirus of claim
 30. 33. An isolated and purified nucleic acid thatencodes the chimeric adenovirus fiber protein of claim
 17. 34. A viraltransfer vector comprising the nucleic acid of claim
 33. 35. A vectorcomprising the nucleic acid of claim 33, wherein said vector is selectedfrom the group consisting of a prokaryotic expression vector, aeukaryotic expression vector, and a recombinant baculovirus.
 36. Achimeric adenovirus fiber protein which comprises a nonnative amino acidsequence in addition to a native fiber amino acid sequence, wherein (1)said nonnative amino acid sequence is a protease recognition sequence ora protein binding sequence, and (2) said nonnative amino acid sequenceis located internally in the chimeric adenovirus fiber protein.
 37. Thechimeric adenovirus fiber protein of claim 36, wherein said native fiberamino acid sequence is a protein binding sequence that has been movedwithin the fiber protein.
 38. The chimeric adenovirus fiber protein ofclaim 36, wherein said nonnative amino acid sequence is in an exposedloop of the chimeric adenovirus fiber protein.
 39. A recombinantadenovirus comprising the chimeric adenovirus fiber protein of claim 36.40. An isolated and purified nucleic acid that encodes the recombinantadenovirus of claim
 39. 41. A method of genetically modifying a cell invitro which comprises introducing into said cell the recombinantadenovirus of claim
 38. 42. An isolated and purified nucleic acid thatencodes the chimeric adenovirus fiber protein of claim
 36. 43. A viraltransfer vector comprising the nucleic acid of claim
 42. 44. A vectorcomprising the nucleic acid of claim 42, wherein said vector is selectedfrom the group consisting of a prokaryotic expression vector, aeukaryotic expression vector, and a recombinant baculovirus.
 45. Achimeric adenovirus fiber protein which comprises a nonnative amino acidsequence in place of a native fiber amino acid sequence, wherein (1)said nonnative amino acid sequence is a protease recognition sequence ora protein binding sequence, and (2) said nonnative amino acid sequenceis located internally in the chimeric adenovirus fiber protein.
 46. Thechimeric adenovirus fiber protein of claim 45, wherein said native fiberamino acid sequence is a protein binding sequence.
 47. The chimericadenovirus fiber protein of claim 45, wherein said nonnative amino acidsequence is in an exposed loop of the chimeric adenovirus fiber protein.48. A recombinant adenovirus comprising the chimeric adenovirus fiberprotein of claim
 45. 49. An isolated and purified nucleic acid thatencodes the recombinant adenovirus of claim
 48. 50. A method ofgenetically modifying a cell in vitro which comprises introducing intosaid cell the recombinant adenovirus of claim
 48. 51. An isolated andpurified nucleic acid that encodes the chimeric adenovirus fiber proteinof claim
 45. 52. A viral transfer vector comprising the nucleic acid ofclaim
 51. 53. A vector comprising the nucleic acid of claim 51, whereinsaid vector is selected from the group consisting of a prokaryoticexpression vector, a eukaryotic expression vector, and a recombinantbaculovirus.
 54. A chimeric adenovirus fiber protein which comprises anonnative amino acid sequence in addition to a native fiber amino acidsequence, wherein (1) said nonnative amino acid sequence is a proteaserecognition sequence or a protein binding sequence, and (2) saidchimeric fiber protein forms a trimer when produced in a mammalian cell.55. The chimeric adenovirus fiber protein of claim 54, wherein saidnonnative amino acid sequence further comprises a trimerization domain.56. The chimeric adenovirus fiber protein of claim 54, wherein saidnonnative amino acid sequence is located internally in the chimericadenovirus fiber protein.
 57. A recombinant adenovirus comprising thechimeric adenovirus fiber protein of claim
 54. 58. An isolated andpurified nucleic acid that encodes the recombinant adenovirus of claim57.
 59. A method of genetically modifying a cell in vitro whichcomprises introducing into said cell the recombinant adenovirus of claim57.
 60. An isolated and purified nucleic acid that encodes the chimericadenovirus fiber protein of claim
 54. 61. A viral transfer vectorcomprising the nucleic acid of claim
 60. 62. A vector comprising thenucleic acid of claim 60, wherein said vector is selected from the groupconsisting of a prokaryotic expression vector, a eukaryotic expressionvector, and a recombinant baculovirus.
 63. A chimeric adenovirus fiberprotein which comprises a nonnative amino acid sequence in place of anative fiber amino acid sequence, wherein (1) said nonnative amino acidsequence is a protease recognition sequence or a protein bindingsequence, and (2) said chimeric fiber protein forms a trimer whenproduced in a mammalian cell.
 64. The chimeric adenovirus fiber proteinof claim 63, wherein said nonnative amino acid sequence furthercomprises a trimerization domain.
 65. The chimeric adenovirus fiberprotein of claim 63, wherein said nonnative amino acid sequence islocated internally in the chimeric adenovirus fiber protein.
 66. Arecombinant adenovirus comprising the chimeric adenovirus fiber proteinof claim
 63. 67. An isolated and purified nucleic acid that encodes therecombinant adenovirus of claim
 66. 68. A method of geneticallymodifying a cell in vitro which comprises introducing into said cell therecombinant adenovirus of claim
 66. 69. An isolated and purified nucleicacid that encodes the chimeric adenovirus fiber protein of claim
 63. 70.A viral transfer vector comprising the nucleic acid of claim
 69. 71. Avector comprising the nucleic acid of claim 69, wherein said vector isselected from the group consisting of a prokaryotic expression vector, aeukaryotic expression vector, and a recombinant baculovirus.
 72. Achimeric adenovirus fiber protein which comprises a nonnative amino acidsequence in addition to a native fiber amino acid sequence, wherein (1)said nonnative amino acid sequence is a protease recognition sequence ora protein binding sequence, and (2) said nonnative amino acid sequenceis joined to said native amino acid sequence by at least one spacersequence.
 73. The chimeric adenovirus fiber protein of claim 72, whereinsaid nonnative amino acid sequence is located internally in the chimericadenovirus fiber protein.
 74. A recombinant adenovirus comprising thechimeric adenovirus fiber protein of claim
 72. 75. An isolated andpurified nucleic acid that encodes the recombinant adenovirus of claim74.
 76. A method of genetically modifying a cell in vitro whichcomprises introducing into said cell the recombinant adenovirus of claim74.
 77. An isolated and purified nucleic acid that encodes the chimericadenovirus fiber protein claim
 72. 78. A viral transfer vectorcomprising the nucleic acid of claim
 77. 79. A vector comprising thenucleic acid of claim 77, wherein said vector is selected from the groupconsisting of a prokaryotic expression vector, a eukaryotic expressionvector, and a recombinant baculovirus.
 80. A chimeric adenovirus fiberprotein which comprises a nonnative amino acid sequence in place of anative fiber amino acid sequence, wherein (1) said nonnative amino acidsequence is a protease recognition sequence or a protein bindingsequence, and (2) said nonnative amino acid sequence is joined to saidnative amino acid sequence by at least one spacer sequence.
 81. Thechimeric adenovirus fiber protein of claim 80, wherein said nonnativeamino acid sequence is located internally in the chimeric adenovirusfiber protein.
 82. A recombinant adenovirus comprising the chimericadenovirus fiber protein of claim
 80. 83. An isolated and purifiednucleic acid that encodes the recombinant adenovirus of claim
 82. 84. Amethod of genetically modifying a cell in vitro which comprisesintroducing into said cell the recombinant adenovirus of claim
 82. 85.An isolated and purified nucleic acid that encodes the chimericadenovirus fiber protein claim
 80. 86. A viral transfer vectorcomprising the nucleic acid of claim
 85. 87. A vector comprising thenucleic acid of claim 85, wherein said vector is selected from the groupconsisting of a prokaryotic expression vector, a eukaryotic expressionvector, and a recombinant baculovirus.
 88. A chimeric adenovirus fiberprotein which comprises a nonnative amino acid sequence in addition to anative fiber amino acid sequence, wherein said nonnative amino acidsequence is a protease recognition sequence or a sequence selected fromthe group consisting of a sequence from a serotype of adenovirus thatdiffers from the serotype of the native fiber amino acid sequence, abispecific protein binding sequence, and a multispecific protein bindingsequence.
 89. A chimeric adenovirus fiber protein which comprises anonnative amino acid sequence in place of a native fiber amino acidsequence, wherein said nonnative amino acid sequence is a proteaserecognition sequence or a sequence selected from the group consisting ofa sequence from a serotype of adenovirus that differs from the serotypeof the native fiber amino acid sequence, a bispecific protein bindingsequence, and a multispecific protein binding sequence.